Microglial activation is a key element in initiating and perpetuating innate immune responses to cerebral ischemia. Microglial responses are characterized as either M1, classical activation (pro-inflammatory), or M2, alternative activation (anti-inflammatory). The two distinct phenotypes of microglia potentially provide a therapeutic avenue that selectively enhances M2 and/or inhibits M1 activation. The mechanism underlying microglial M1/M2 activation after stroke has not been explored. Studies of peripheral inflammation suggested that interferon regulatory factor 5 (IRF5) and IRF4 are key determinants in mediating macrophage M1/M2 polarization. IRF5 and IRF4 function through TLR4- MyD88-IRF5 and IL4R-Jmjd3-IRF4 pathways to regulate macrophage M1/M2 phenotype respectively. It is suggested that the two pathways act synergistically to mediate M1/M2 polarization. We hypothesize that the IRF5-IRF4 regulatory axis balances the TLR4-MyD88-IRF5 and IL4R-Jmjd3-IRF4 pathways to direct microglial M1/M2 polarization after stroke, and that manipulation of the IRF5-IRF4 regulatory axis confers neuroprotection against ischemia. Inducible conditional knockout (ICKO) and a bone marrow chimera mouse model will be utilized to study the IRF5-IRF4 regulatory axis specifically in microglia or in infiltrating peripheral leukocytes afer stroke. Aim 1 will study the role of the IRF5-IRF4 regulatory axis selectively in microglia after ischemic injury. Using a chronic ICKO model in which IRF5/IRF4 is selectively lost in microglia but intact in peripheral leukocytes, protein over-expression and knockdown, and microglia-neuron co- culture, we will test if manipulation of the IRF5-IRF4 regulatory axis can switch microglial phenotype and reduce ischemic injury after experimental stroke. Aim 2 will investigate whether the IRF5-IRF4 regulatory axis in infiltrating peripheral cells plays an important role in mediating post-ischemic brain injury. Bone marrow chimeras and an acute IRF5/IRF4 ICKO mouse model will be employed. Aim 3 will examine if age-related differences exist in the IRF5-IRF4 regulatory axis after stroke. The IRF5/IRF4 ICKO model will be utilized to examine the IRF5/IRF4 signaling in aged mice. Manipulation of the IRF5- IRF4 regulatory axis in microglia/infiltrating leukocytes may help limit ischemic injury and promote tissue repair after stroke. The study has high translational value and will hopefully identify new biological targets for therapeutic intervention for patients with stroke.